Tryptase polypeptide and uses thereof

ABSTRACT

The present invention provides a purified expressed δ tryptase polypeptide or fragment or analogue thereof, in particular, the human δ tryptase polypeptide or fragment or analogue thereof. The present invention also provides variants of such polypeptides. The invention further relates to methods of diagnosing disease states based on expression of these polypeptides and methods of screening for compounds that interact, bind and/or modulate the activity of the polypeptides.

TECHNICAL FIELD

The present invention relates to the expressed delta (δ) tryptasepolypeptde, in particular the human δ tryptase protein. The presentinvention also relates to uses of this polypeptide, in particular fordiagnosing disease states and screening for δ tryptase modulator andinhibitor compounds.

BACKGROUND OF THE INVENTION

Mast cells are highly granulated, tissue-resident, effector cells of theimmune system. They may be activated via their high affinity IgEreceptors or by a number of alternate mechanisms. Following activationthey secrete a variety of preformed mediators including histamine,proteoglycans, and a range of serine proteases that are active atneutral pH. Two major families of these proteases have been identified:chymases and tryptases, and they represent the major protein componentof the mast cell.

A role has been proposed for mast cell tryptases in the development of anumber of inflammatory diseases, including diseases of the respiratorytract, such as asthma and allergic rhinitis, rheumatoid arthritis, andinflammatory bowel disease. Despite the underlying causes of thesediseases not being fully understood, it is known that the number of mastcells is increased in the airways of patients with asthma, and in thesynovial tissue of patients with rheumatoid arthritis.

Accordingly, there is a clear need to elucidate the role of mast cellsin inflammatory disease phenotypes and to develop suitable agents forthe prevention and/or inhibition of mast cell-mediated inflammation.

There is a wealth of evidence indicating that many mast cell productsare pro-inflammatory. However, recent reports suggest that tryptase maybe one of the most important in the development of asthma andinflammatory bowel disorders. Tryptases have been shown to inactivatevasoactive intestinal peptide (VIP), one of only two naturally occurringbronchodilators expressed in the airway, and to induce smooth musclecell mitosis and hyper-reactivity. More importantly tryptase inhibitorsinhibit allergen-induced airway hyperreactivity and markers ofinflammation. Tryptase also induces IL-8 secretion from airwayepithelial cells, so promoting airway inflammation.

Tryptases have thus become the focus of considerable attention, andthere exists a need for the elucidation of effective modulators andinhibitors of tryptases which may be used in treating or preventing mastcell-mediated inflammatory diseases.

A major impediment to determining the role of tryptases, and thereforeto developing effective mdoulators, inhibitors and treatments, has beenthe confusion over how many different functional human tryptases areexpressed. A number of tryptase genes are known to be grouped onchromosome 16, mapping to 16p13.3. These include the gene encoding βItryptase and its allelic αII tryptase, the allelic genes encoding βIIand βIII tryptase, two allelic variants of a transmembrane tryptasecalled gamma (γ) tryptase, and two allelic variants of another tryptaseoriginally named “mMCP-7-like” (Pallaoro et al., 1999; Caughey et al.,2000). Of these, cDNAs have been cloned for all loci except for thatencoding “mMCP-7-like” tryptase. Recently the cloning of a moredistantly related member, epsilon (ε) tryptase, which is approximately40% similar to the α/β tryptases has also been described (Wong et al.,2001).

The mMCP-7-like tryptase was so named due to homology between its fifthexon and the to murine tryptase mouse mast cell protease (mMCP)-7(Pallaoro et al., 1999; McNeil et al., 1992). Recently it has beenreported (Min et al., 2001) that the mMCP-7-like gene is nottranscribed. Based on the examination of a number of human tissues andcell lines for transcription of the mMCP-7-like gene, Min et al.reported that mRNA is absent and concluded that the mMCP-7-like gene isa pseudogene. However the present inventors have surprisingly discoveredthat the human mMCP-7-like tryptase is indeed expressed and have namedthe gene, and its polypeptide product delta (δ) tryptase.

SUMMARY OF THE INVENTION

According to a first embodiment of the present invention there isprovided a purified, expressed δ tryptase polypeptide or fragment oranalogue thereof. Preferably, the expressed δ tryptase polypeptde is thehuman δ tryptase protein. More preferably, the expressed human δtryptase protein has the amino acid sequence as set forth in SEQ ID NO:1or SEQ ID NO:2, or the amino acid sequence as set forth in SEQ ID NO:1or SEQ ID NO:2 including one or more conservative amino acidsubstitutions.

According to a second embodiment of the present invention there isprovided a purified, expressed variant of the δ tryptase polypeptide ofthe first embodiment, or a fragment or analogue of this variant.Preferably, the variant δ tryptase polypeptide is the product ofalternative splicing of the primary RNA transcript. More preferably thevariant polypeptide has the amino acid sequence as set forth in SEQ IDNO:3.

In a third embodiment the present invention provides a recombinant hostcell expressing the polypeptide or fragment or analogue thereof of thefirst or second embodiment.

In a fourth embodiment the present invention provides an antibody thatselectively binds to the polypeptide or fragment or analogue thereof ofthe first or second embodiment.

In a fifth embodiment the present invention provides a method ofidentifying a compound that interacts with the polypeptide or fragmentor analogue thereof of the first or second embodiment, the methodcomprising the steps of:

(a) contacting a candidate compound with the polypeptide or fragment oranalogue thereof of the first or second embodiment under conditionssuitable to enable interaction of the candidate compound to thepolypeptide or fragment or analogue thereof; and

(b) assaying for activity of the polypeptide or fragment or analoguethereof.

Preferably, assaying for activity of the polypeptide or fragment oranalogue thereof comprises adding a labelled substrate and measuring achange in the labelled substrate.

According to a sixth embodiment of the present invention there isprovided a method of identifying a compound that binds to thepolypeptide or fragment or analogue thereof of the first or secondembodiment, the method comprising the steps of:

(a) contacting a candidate compound with the polypeptide or fragment oranalogue thereof of the first or second embodiment; and

(b) assaying for the formation of a complex between the candidatecompound and the polypeptide or fragment or analogue thereof.

Preferably the assay for the formation of a complex is a competitivebinding assay or a two-hybrid assay.

According to a seventh embodiment of the present invention there isprovided a method of screening for a compound that modulates theactivity of the polypeptide or fragment or analogue thereof of the firstor second embodiment, the method comprising the steps of:

(a) contacting the polypeptide or fragment or analogue thereof of thefirst or second embodiment with a candidate compound under conditionssuitable to enable interaction of the candidate compound to thepolypeptide; and

(b) assaying for activity of the polypeptide of the first or secondembodiment.

Preferably, assaying for activity of the polypeptide comprises adding alabelled substrate and measuring a change in the labelled substrate.

Preferably the modulation of activity is an inhibition of activity ofthe polypeptide or fragment or analogue thereof of the first or secondembodiment.

According to an eighth embodiment of the present invention there isprovided a method of diagnosing a disease state, or predisposition to adisease state, in a subject, the method comprising the steps of:

(a) isolating a biological sample from the subject; and

(b) assaying for expression of the polypeptide or fragment or analoguethereof of the first or second embodiment in the sample.

Preferably, assaying for the expression of the polypeptide or fragmentor analogue thereof comprises contacting the biological sample with acompound capable of interacting with the polypeptide such that theinteraction can be detected.

Preferably the compound capable of interacting with the polypeptide orfragment or analogue thereof is an anti-δ tryptase antibody.

Preferably the disease state is an inflammatory disease. Morepreferably, the disease is a mast-cell associated inflammatory disease.Most preferably the inflammatory disease is selected from the groupconsisting of: asthma, allergic rhinitis, urticaria, angioedema,eczematous anaphylaxis, dermatitis such as atopic dermatitis,hyperproliferative skin disease, peptic ulcers, inflammatory boweldisorder, ocular and vernal conjunctivitis, rheumatoid arthritis, andinflammatory skin conditions.

According to a ninth embodiment of the present invention there isprovided a method of identifying an agent which is an inhibitor of mastcell-mediated inflammation, the method comprising contacting a cell orcell extract with the agent, determining whether there is a change inthe activity of a δ tryptase polypeptide or fragment or analoguethereof, and thereby determining whether the agent is an inhibitor ofmast cell-mediated inflammation.

According to a tenth embodiment of the present invention there isprovided a method of identifying an agent suitable for use in thetreatment or prevention of a mast cell-mediated inflammatory diseasestate in a subject, the method comprising isolating a biological samplefrom the subject, contacting the sample with a candidate agent,determining whether there is a change in the activity a δ tryptasepolypeptide or fragment or analogue thereof in the sample, and therebydetermining whether the agent is suitable for use in the treatment ofthe disease state.

Preferably the inflammatory disease is selected from the groupconsisting of: asthma, allergic rhinitis, urticaria, angioedema,eczematous anaphylaxis, dermatitis such as atopic dermatitis,hyperproliferative skin disease, peptic ulcers, inflammatory boweldisorder, ocular and vernal conjunctivitis, rheumatoid arthritis, andinflammatory skin conditions.

In an eleventh embodiment the present invention provides compoundsidentified by the methods of the fifth, sixth, seventh, ninth and tenthembodiments.

In a twelfth embodiment the present invention provides a method fortreating or preventing a disease state in a subject, the methodcomprising administering to the subject a therapeutically effectiveamount of a compound identified by the method of any one of the fifth,sixth, seventh, ninth and tenth embodiments.

According to a thirteenth embodiment of the present invention there isprovided a kit comprising the polypeptide or fragment or analoguethereof of the first or second embodiment. Alternatively, or inaddition, the kit may contain an antibody of the fourth embodiment.Preferably the kit is used for carrying out the methods of the fifth, tothe tenth embodiments.

BRIEF DESCRIPTION OF THE DRAWINGS

Preferred embodiments of the present invention will now be described, byway of example only, with reference to the accompanying drawings.

FIG. 1. RT-PCR amplification of the transcript for δ tryptase. A).RT-PCR of total RNA isolated from the HMC-1 cell line using two primerpair combinations, F1/R1 and F2/R2. GAPDH was amplified as a control.The correct sized band (832 bp) for δ tryptase was only generated usingthe F1/R1 primer pair. B). PCR amplification using the purified 832 bpband from FIG. 1A as the DNA template, and the nested primer set NF1/NR1to generate the expected 698 bp product.

FIG. 2. A). The cDNA and putative amino acid sequences of δII tryptase(SEQ ID NO:1). The δII cDNA sequence matched the putative exon sequenceof the mMCP-7-like II gene. The 81 cDNA (not shown in FIG. 2) matchedthe exonic sequence of the partial mMCP-7-like I gene. Consistent withthe published gene sequences, there were two nucleotide differencesbetween the two cDNA sequences; G²¹⁶ (δII cDNA) to A (δI cDNA)(nucleotide numbering starts from the translation initiation codon), andG²²⁶ (δII cDNA) to A (δI cDNA). Only the second of these differencesresults in an amino substitution (Val in δII to Met in δI). Actualnucleotide sequence of cloned RT-PCR product is shown in bold lower caselettering. The location of the forward (NF1) and reverse (NR1) primersare indicated by arrows. The first amino acid of the mature enzyme isitalicized and in bold. The three members of the catalytic triad, His,Asp and Ser, are in capitals and underlined. Nucleotide numbering beginsfrom the translation initiation codon (Met). Amino acid numbering beginsfrom the first residue of the mature enzyme (Ile). The position of theforward (

) and reverse (

) primers, and the Taqman probe (═) for RTQ-RTPCR are indicated. B). Theamino acid sequence of a variant δ tryptase polypeptide. This variant isthe product of alternative splicing which results in the excision of 27nulceotides from the beginning of exon 4, and thus the deletion of 9amino acids from the polypeptide when compared to the full length δtryptase polypeptide. The location of the 9 amino acids present in thefull length polypeptide but missing in the variant polypeptide isindicated by a vertical arrow (↓).

FIG. 3. Amino acid sequences of δI tryptase and δII tryptase compared tothat of tryptases ΕI, αII, βI, βII and βIII. A dash (-) indicates thepresence of an identical amino acid. Numbering begins at the firstresidue of the mature enzyme, which is indicated by an arrow (▾). Theseven loops comprising the substrate binding cleft are boxed andlabelled A,B,C,D,1,2,and 3. The H, D and S of the catalytic triad aremarked with a hash (#). The premature termination codons of the δtryptases are marked with an X. The peptide sequence used as theimmunogen for anti δ tryptase is underlined ( . . . ).

FIG. 4. δ tryptase is transcribed in various tissues. The relativeabundance of δ tryptase mRNA (normalised with respect to β-actin mRNA)in a range of human tissues was determined using RTQ-RTPCR. The datarepresents the mean (± SD) from a single experiment. All samples weretested in triplicate and have been tested in at least two independentexperiments, except for colon RNA which was tested once. NTC=no templatecontrol.

FIG. 5. Affinity purified anti-δ tryptase antibody recognises rδtryptase but not rβII tryptase. Approximately 0.5 μg of purified rδtryptase and 1 μg of rβII tryptase (Promega) were separated on a 10% SDSpolyacrylamide gel and transferred to a PVDF membrane, blocked, andincubated with affinity purified δ tryptase anti-peptide antibody (1μg/ml) (FIG. 5A). Replicate blots were probed with normal rabbit IgG (1μg/ml) (data not shown), and with the mouse monoclonal anti-tryptaseantibody AA1 (1/200) (FIG. 5B)

FIG. 6. δ tryptase protein is expressed in human colon tissue. Sectionsof human colon were stained immunohistochemically using anti-δ tryptaseIg (A,B, and E)-, the monoclonal anti-tryptase antibody AA1 (D), andnormal rabbit IgG (C and F). Panels B and C, and panels D through F areserial sections. Arrows in panels D and E indicate the location of acell that is stained with both anti-δ tryptase and the AA1 antibody.

FIG. 7. rδ tryptase cleaves a trypsin-sensitive substrate. Reverse-phaseHPLC of D-Ile-Phe-Lys pNA cleavage products when digested with A) bufferalone, B) rβII tryptase, C) native lung tryptase, D) rαII tryptase E) rβtryptase, or F) pro-rδ tryptase. Approximately 2 μg of each enzyme wasused to digest 10 μg of substrate in a 50 μl reaction. Liberated pNA (▾)and peptide (

) moieties are indicated. The large peak at the right of each panel (5.6mins) represents undigested substrate. Samples were tested in duplicateand representative chromatograms are shown.

DEFINITIONS

As used herein the term “polypeptide” means a polymer made up of aminoacids linked together by peptide bonds.

The term “purified” means that the material in question has been removedfrom its host, and associated impurities reduced or eliminated.Essentially, it means an object species is the predominant speciespresent (ie., on a molar basis it is more abundant than any otherindividual species in the composition), and preferably a substantiallypurified fraction is a composition wherein the object species comprisesat least about 30 percent (on a molar basis) of all macromolecularspecies present. Generally, a substantially pure composition willcomprise more than about 80 to 90 percent of all macromolecular speciespresent in the composition. Most preferably, the object species ispurified to essential homogeneity (contaminant species cannot bedetected in the composition by conventional detection methods) whereinthe composition consists essentially of a single macromolecular species.

The term “fragment” refers to a polypeptide molecule that is aconstituent of the full-length δ tryptase polypeptide and possessesqualitative biological activity in common with the full-length δtryptase polypeptide. The fragment may be derived from the full-length δtryptase polypeptide or alternatively may be synthesised by some othermeans, for example chemical synthesis.

The term “analogue” as used herein with reference to a polypeptide meansa polypeptide which is a derivative of the polypeptide of the invention,which derivative comprises addition, deletion, substitution of one ormore amino acids, such that the polypeptide retains substantially thesame function as the δ tryptase polypeptide identified above.

The term “variant” as used herein refers to a polypeptide which isproduced from the nucleic acid encoding δ tryptase, but differs from thewild type δ tryptase in that it is processed differently such that ithas an altered amino acid sequence. For example a variant may beproduced by an alternative splicing pattern of the primary RNAtranscript to that which produces wild type δ tryptase.

The term “conservative amino acid substitution” as used herein refers toa substitution or replacement of one amino acid for another amino acidwith similar properties within a polypeptide chain (primary sequence ofa protein). For example, the substitution of the charged amino acidglutamic acid (Glu) for the similarly charged amino acid aspartic acid(Asp) would be a conservative amino acid substitution.

The term “modulator” as used herein refers to any compound capable ofinteracting with a δ tryptase polypeptide, or fragment, analogue orvariant thereof, so as to alter the expression, catalytic activity, orother function of the δ tryptase polypeptide, or fragment, analogue orvariant thereof. A modulator may be, for example, an agonist, antagonistor inhibitor of the δ tryptase polypeptide, or fragment, analogue orvariant thereof. Modulators may include antibodies, low molecular weightpeptides, nucleic acids or non-proteinaceous organic molecules, forexample.

As used herein the term “treatment”, refers to any and all uses whichremedy a disease state or symptoms, prevent the establishment ofdisease, or otherwise prevent, hinder, retard, or reverse theprogression of disease or other undesirable symptoms in any waywhatsoever.

As used herein the term “therapeutically effective amount” includeswithin its meaning a non-toxic but sufficient amount of an agent orcompound to provide the desired therapeutic effect. The exact amountrequired will vary from subject to subject depending on factors such asthe species being treated, the age and general condition of the subject,the severity of the condition being treated, the particular agent beingadministered and the mode of administration and so forth. Thus, it isnot possible to specify an exact “effective amount”. However, for anygiven case, an appropriate “effective amount” may be determined by oneof ordinary skill in the art using only routine experimentation.

In the context of this specification, the term “comprising” means“including principally, but not necessarily solely”. Furthermore,variations of the word “comprising”, such as “comprise” and “comprises”,have correspondingly varied meanings.

BEST MODE OF PERFORMING THE INVENTION

The present invention is based on the inventors' surprising finding thatthe human δ tryptase protein is not a pseudogene, but rather isexpressed in a number of human tissues.

The inventors have also generated an antibody directed against the humanδ tryptase protein and a recombinant δ tryptase protein, displayingproteolytic activity, in a bacterial expression system. The recombinantδ tryptase generated possesses full catalytic activity.

δ Tryptase Polypeptides

Accordingly, an embodiment of the present invention provides theisolated, expressed δ tryptase polypeptide, or fragment or analoguethereof. Particularly, the δ tryptase polypeptide of the presentinvention is the human δ tryptase protein, having the amino acidsequence set forth in SED ID:1 (δI) or SEQ ID:2 (δII). It will beappreciated that this amino acid sequence may include one or moreconservative amino acid substitutions such that although the primarysequence of the polypeptide is altered, the activity of the polypeptideis retained. The present invention also relates to variants of the δtryptase polypeptide. In a preferred embodiment, the variant δ tryptasepolypeptide is a polypeptide having the amino acid sequence as set forthin SEQ ID NO:3. The variant polypeptide sequence depicted in SEQ ID NO:3is generated by alternative splicing of the primary δ tryptasetranscript, such that the initial 27 nucleotides of exon 4 are excised,resulting in a polypeptide 9 amino acids shorter than the mature fulllength human δ tryptase.

The in vivo expression of human δ tryptase transcript has been observedin a wide variety of tissues, including lung, heart, stomach, spleen,skin, and colon. The δ tryptase transcript appears to be most abundantin the lung and colon but is also expressed in the heart and stomach.The present application provides the first evidence that the human δtryptase gene is expressed at the transcriptional level.

The human δ tryptase polypeptide of the present invention is expressedin a number of tissues including colon, lung, heart and synovial tissue.In the colon, the vast majority of δ tryptase-positive cells were foundto be in the mucosal compartment, with only one or two individual δtryptase-positive cells located in the submucosa or muscle layers. Thissuggests that δ tryptase may be useful as marker of a new mast cellphenotype that is more common in the mucosa.

Detection of the δ tryptase transcript in the HMC-1 cell line indicatesthat its expression may be primarily restricted to mast cells. Howeverimmunohistochemistry results suggest that δ tryptase may be expressed byother cells in addition to mast cells and/or that discordant mast cellphenotypes may exist characterised by discordant expression of δtryptase and the α/β tryptases.

The mature human δ tryptase enzyme is 40 amino acids shorter than thewell described α/β family of human tryptases. However the catalytictriad, essential for proteolytic activity, remains intact. The substratebinding cleft of tryptases comprises seven major loops in thepolypeptide chain, named A, B, C, D, 3, 1 and 2 in order from the Nterminus (see FIG. 3). The loss of the terminal 40 amino acids in δtryptase means that loop 2 (residues 211-218 in the α/β tryptases) isnot present in δ tryptase. This loop forms part of the S1, S2 and S3sites of the enzyme and even single amino acid changes within this loopis known to alter the substrate specificity of tryptases (Huang et al.1999). Consistent with this, a recombinant form of δ tryptase, expressedin bacterial cells, has been shown to differ from βII tryptase in itsability to cleave a panel of three substrates. It appears that thesubstrate specificity of δ tryptase is likely to be quite different fromthat of previously characterised tryptases.

Huang et al. (2000) have reported that two mouse tryptases, mMCP-6 andmMCP-7 could form enzymatically-active heterotypic tetramers. Further,Harris et al. (2001) have suggested that residues on neighbouringtryptase monomers acted together within the tetramer framework to formparts of the extended substrate recognition site. Taken together theseresults suggest that even if its proteolytic efficiency is low, δtryptase may modulate the activity of other tryptases by formingheterotypic tetramers.

In vitro detection of the polypeptides or fragments or analogues thereofof the present invention may be achieved using a variety of techniquesincluding ELISA (enzyme linked immunosorbent assay), Western blotting,immunoprecipitation and immunofluorescence. Such techniques are commonlyused by those of skill in the art. Similarly, suitable techniques of thein vivo detection of the polypeptide, or fragments or analogues thereof,including immunohistochemistry using a labelled anti-δ tryptaseantibody, will be readily understood by persons skilled in the art.

In accordance with the present invention, fusion proteins may also beengineered to improve characteristics of the δ tryptase polypeptides, orfragments or analogues thereof, of the present invention. For example, aregion of additional amino acids, particularly charged amino acids, maybe added to the N-terminus of the δ tryptase polypeptides to improvestability during purification from a host cell. Alternatively, peptidemoieties may be added to the polypeptide to facilitate purification.Such regions may be removed prior to final preparation of thepolypeptide. The addition of peptide moieties to facilitate handling ofpolypeptides are routine techniques well known to those of skill in theart.

Uses of the δ Tryptase Polypeptides

Antibodies

The present invention provides antibodies that selectively bind to the δtryptase of the present invention, as well as fragments and analoguesthereof. Suitable antibodies include, but are not limited to polyclonal,monoclonal, chimeric, humanised, single chain, Fab fragments, and an Fabexpression library. Antibodies of the present invention may act asagonists or antagonists of δ tryptase polypeptides, or fragments oranalogues thereof.

Preferably antibodies are prepared from discrete regions or fragments ofthe tryptase polypeptide, in particular those involved in conferringprotease activity and/or partner or substrate binding. An antigenic δtryptase polypeptide contains at least about 5, and preferably at leastabout 10, amino acids.

Methods for the generation of suitable antibodies will be readilyappreciated by those skilled in the art. For example, an anti-δ tryptasemonoclonal antibody, typically containing Fab portions, may be preparedusing the hybridoma technology described in Antibodies-A LaboratoryManual, Harlow and Lane, eds., Cold Spring Harbor Laboratory, N.Y.(1988).

In essence, in the preparation of monoclonal antibodies directed towardδ tryptase polypeptide, fragment or analogue, thereof, any techniquethat provides for the production of antibody molecules by continuouscell lines in culture may be used. These include the hybridoma techniqueoriginally developed by Kohler et al., Nature, 256:495-497 (1975), aswell as the trioma technique, the human B-cell hybridoma technique[Kozbor et al., Immunology Today, 4:72 (1983)], and the EBV-hybridomatechnique to produce human monoclonal antibodies [Cole et al., inMonoclonal Antibodies and Cancer Therapy, pp. 77-96, Alan R. Liss, Inc.,(1985)]. Immortal, antibody-producing cell lines can be created bytechniques other than fusion, such as direct transformation of Blymphocytes with oncogenic DNA, or transfection with Epstein-Barr virus.See, e.g., M. Schreier et al., “Hybridoma Techniques” (1980); Hammerlinget al., “Monoclonal Antibodies and T-cell Hybridomas” (1981); Kennett etal., “Monoclonal Antibodies” (1980).

In summary, a means of producing a hybridoma from which the monoclonalantibody is produced, a myeloma or other self-perpetuating cell line isfused with lymphocytes obtained from the spleen of a mammalhyperimmunised with a recognition factor-binding portion thereof, orrecognition factor, or an origin-specific DNA-binding portion thereof.Hybridomas producing a monoclonal antibody useful in practicing thisinvention are identified by their ability to immunoreact with thepresent recognition factor and their ability to inhibit specifiedtranscriptional activity in target cells.

A monoclonal antibody useful in practicing the present invention can beproduced by initiating a monoclonal hybridoma culture comprising anutrient medium containing a hybridoma that secretes antibody moleculesof the appropriate antigen specificity. The culture is maintained underconditions and for a time period sufficient for the hybridoma to secretethe antibody molecules into the medium. The antibody-containing mediumis then collected. The antibody molecules can then be further isolatedby well-known techniques.

Similarly, there are various procedures known in the art which may beused for the production of polyclonal antibodies to δ tryptase, orfragments or analogues thereof. For the production of δ tryptasepolyclonal antibody, various host animals can be immunized by injectionwith the δ tryptase polypeptide, or a fragment or analogue thereof,including but not limited to rabbits, mice, rats, sheep, goats, etc.Further, the δ tryptase polypeptide or fragment or analogue thereof canbe conjugated to an immunogenic carrier, e.g., bovine serum albumin(BSA) or keyhole limpet hemocyanin (KLH). Also, various adjuvants may beused to increase the immunological response, including but not limitedto Freund's (complete and incomplete), mineral gels such as aluminiumhydroxide, surface active substances such as lysolecithin, pluronicpolyols, polyanions, peptides, oil emulsions, keyhole limpethemocyanins, dinitrophenol, and potentially useful human adjuvants suchas BCG (bacille Calmette-Guerin) and Corynebacterium parvum.

Screening for the desired δ tryptase antibody can also be accomplishedby a variety of techniques known in the art. Assays for immunospecificbinding of antibodies may include, but are not limited to,radioimmunoassays, ELISAs (enzyme-linked immunosorbent assay), sandwichimmunoassays, immunoradiometric assays, gel diffusion precipitationreactions, immunodiffusion assays, in situ immunoassays, Western blots,precipitation reactions, agglutination assays, complement fixationassays, immunofluorescence assays, protein A assays, andimmunoelectrophoresis assays, and the like (see, for example, Ausubel etal., eds, 1994, Current Protocols in Molecular Biology, Vol. 1, JohnWiley & Sons, Inc., New York). Antibody binding may be detected byvirtue of a detectable label on the primary anti δ tryptase antibody.Alternatively, the anti δ tryptase antibody may be detected by virtue ofits binding with a secondary antibody or reagent which is appropriatelylabelled. A variety of methods are known in the art for detectingbinding in an immunoassay and are within the scope of the presentinvention.

Antibodies of the present invention can be used in diagnostic methodsand kits that are well known to those of ordinary skill in the art todetect qualitatively or quantify δ tryptase in a body fluid or tissue,and results from these tests can be used to diagnose or determinepredisposition to an inflammatory disease in a subject.

The antibody (or fragment thereof) raised against δ tryptase or afragment or analogue thereof has binding affinity for δ tryptase.Preferably, the antibody (or fragment thereof) has binding affinity oravidity greater than about 10⁵ M⁻¹, more preferably greater than about10⁶ M⁻¹, more preferably still greater than about 10⁷ M⁻¹ and mostpreferably greater than about 10⁸ M⁻¹.

In terms of obtaining a suitable amount of an antibody according to thepresent invention, one may manufacture the antibody(s) using batchfermentation with serum free medium. After fermentation the antibody maybe purified via a multistep procedure incorporating chromatography andviral inactivation/removal steps. For instance, the antibody may befirst separated by Protein A affinity chromatography and then treatedwith solvent/detergent to inactivate any lipid enveloped viruses.Further purification, typically by anion and cation exchangechromatography may be used to remove residual proteins,solvents/detergents and nucleic acids. The purified antibody may befurther purified and formulated into 0.9% saline using gel filtrationcolumns. The formulated bulk preparation may then be sterilised andviral filtered and dispensed.

Modulator and Inhibitor Compounds

In addition to specific anti-δ tryptase antibodies, the polypeptide ofthe present invention, and fragments and analogues thereof areparticularly useful for the screening and identification of compoundsand agents that interact with δ tryptase. In particular, desirablecompounds are those that modulate the activity of δ tryptase. Suchcompounds may modulate by activating, increasing, inhibiting orpreventing δ tryptase activity. Suitable compounds may exert theireffect on δ tryptase by virtue of either a direct (for example binding)or indirect interaction.

Compounds which bind, or otherwise interact with δ tryptase, andspecifically compounds which modulate the activity of δ tryptase, may beidentified by a variety of suitable methods. Interaction and/or bindingmay be determined using standard competitive binding assays ortwo-hybrid assay systems.

For example, the two-hybrid assay is a yeast-based genetic assay system(Fields and Song, 1989) typically used for detecting protein-proteininteractions. Briefly, this assay takes advantage of the multi-domainnature of transcriptional activators. For example, the DNA-bindingdomain of a known transcriptional activator may be fused to a δ-tryptaseprotein, or fragment or analogue thereof, and the activation domain ofthe transcriptional activator fused to a candidate protein. Interactionbetween the candidate protein and the δ-tryptase, or fragment oranalogue thereof, will bring the DNA-binding and activation domains ofthe transcriptional activator into close proximity. Interaction can thusbe detected by virtue of transcription of a specific reporter geneactivated by the transcriptional activator.

Alternatively, affinity chromatography may be used to identify δtryptase binding partners. For example, a δ tryptase polypeptide, orfragment or analogue thereof, may be immobilised on a support (such assepharose) and cell lysates passed over the column. Proteins binding tothe immobilised δ tryptase polypeptide, fragment or analogue can then beeluted from the column and identified. Initially such proteins may beidentified by N-terminal amino acid sequencing for example.

Alternatively, in a modification of the above technique, a fusionprotein may be generated by fusing a δ tryptase polypeptide, fragment oranalogue to a detectable tag, such as alkaline phosphatase, and using amodified form of immunoprecipitation as described by Flanagan and Leder(1990).

Methods for detecting compounds that modulate δ tryptase activity mayinvolve combining δ tryptase with a candidate compound and a suitablelabelled substrate and monitoring the effect of the compound on δtryptase by changes in the substrate (may be determined as a function oftime). Suitable labelled substrates include those labelled forcolourimetric, radiometric, fluorimetric or fluorescent resonance energytransfer (FRET) based methods, for example. Alternatively, compoundsthat modulate the activity of δ tryptase may be identified by comparingthe catalytic activity of δ tryptase in the presence of a candidatecompound with the catalytic activity of δ tryptase in the absence of thecandidate compound.

The present invention also contemplates compounds which may exert theirmodulatory effect on δ tryptase by altering expression of the protein.In this case, such compounds may be identified by comparing the level ofexpression of δ tryptase in the presence of a candidate compound withthe level of expression of δ tryptase in the absence of the candidatecompound.

δ tryptase polypeptides and appropriate fragments and analogues can beused in high-throughput screens to assay candidate compounds for theability to bind to, or otherwise interact with δ tryptase. Thesecandidate compounds can be further screened against functional δtryptase to determine the effect of the compound on δ tryptase activity.

It will be appreciated that the above described methods are merelyexamples of the types of methods which may be employed to idenfitycompounds that are capable of interacting with, or modulating theactivity of, the δ tryptase polypeptides, and fragments and analoguesthereof, of the present invention. Other suitable methods will be knownto persons skilled in the art and are within the scope of the presentinvention.

By the above methods, compounds can be identified which either activate(agonists) or inhibit (antagonists) δ tryptase activity. Such compoundsmay be, for example, antibodies, low molecular weight peptides, nucleicacids or non-proteinaceous organic molecules.

Potential modulators of δ tryptase activity, for screening by the abovemethods, may be generated by a number of techniques known to thoseskilled in the art. For example, various forms of combinatorialchemistry may be used to generate putative non-peptide modulators.Additionally, techniques such as nuclear magnetic resonance (NMR) and Xray crystallography, may be used to model the structure of δ tryptasepolypeptides, fragments and analogues and computer predictions used togenerate possible modulators (in particular inhibitors) that will fitthe shape of the substrate binding cleft of the tryptase.

Disease Treatment and Diagnosis

Compounds identified by the above methods may be useful as therapeuticagents. These compounds find use, for example, in treating or preventinga disease state in a subject, by administering a therapeuticallyeffective amount of such a compound to the subject. Accordingly,pharmaceutically useful compositions comprising modulators of δ-tryptaseactivity for use in treating or preventing disease states associatedwith tryptase activity are contemplated. Suitable compositions may beformulated according to known methods such as, for example, by theadmixture of a pharmaceutically acceptable carrier and an effectiveamount of the modulator.

The δ tryptase polypeptide of the present invention, fragments andanalogues thereof, and anti δ tryptase antibodies are also particularlyuseful for determining the presence of a disease state in a subject, orthe predisposition of a subject to a disease state, the disease statebeing one that is associated with tryptase activity and/or mast cellactivation. The δ tryptase polypeptide of the present invention, andfragments and analogues thereof, can be used to identify compounds thatmodulate catalytic activity of the δ tryptase polypeptide either in itsnatural state or in an altered form that causes a specific disease orpathology.

Accordingly, the present invention provides suitable methods fordetermining the expression of δ tryptase transcript in biologicalsamples (including cells and tissues), such as reverse transcriptionpolymerase chain reaction (RT-PCR) and real time quantitative (RTQ)RT-PCR. The invention also provides methods for detecting the expressionof δ tryptase polypeptide (as described above).

Diseases which the polypeptides and methods of the present invention areparticularly useful for diagnosing (presence or predisposition in asubject) are inflammatory diseases and disorders associated withhypersensitivity reactions. In particular, diseases characterised bymast cell-mediated inflammation are contemplated. These includeinflammatory disorders of the respiratory tract, inflammatory skinconditions and other inflammatory disorders.

In particular, suitable diseases and disorders include asthma, allergicrhinitis, urticaria and angioedema, eczematous anaphylaxis, dermatitissuch as atopic dermatitis, hyperproliferative skin disease, pepticulcers, inflammatory bowel disorder, ocular and vernal conjunctivitis,rheumatoid arthritis, and inflammatory skin conditions.

Modulator and inhibitor compounds and agents of the present inventionmay be administered as compositions either therapeutically orpreventively. In a therapeutic application, compositions areadministered to a patient already suffering from a disease, in an amountsufficient to cure or at least partially arrest the disease and itscomplications. The composition should provide a quantity of the compoundor agent sufficient to effectively treat the patient.

The therapeutically effective dose level for any particular patient willdepend upon a variety of factors including: the disorder being treatedand the severity of the disorder; activity of the compound or agentemployed; the composition employed; the age, body weight, generalhealth, sex and diet of the patient; the time of administration; theroute of administration; the rate of sequestration of the agent orcompound; the duration of the treatment; drugs used in combination orcoincidental with the treatment, together with other related factorswell known in medicine.

One skilled in the art would be able, by routine experimentation, todetermine an effective, non-toxic amount of agent or compound whichwould be required to treat applicable diseases.

Generally, an effective dosage is expected to be in the range of about0.0001 mg to about 1000 mg per kg body weight per 24 hours; typically,about 0.001 mg to about 750 mg per kg body weight per 24 hours; about0.01 mg to about 500 mg per kg body weight per 24 hours; about 0.1 mg isto about 500 mg per kg body weight per 24 hours; about 0.1 mg to about250 mg per kg body weight per 24 hours; about 1.0 mg to about 250 mg perkg body weight per 24 hours. More typically, an effective dose range isexpected to be in the range about 1.0 mg to about 200 mg per kg bodyweight per 24 hours; about 1.0 mg to about 100 mg per kg body weight per24 hours; about 1.0 mg to about 50 mg per kg body weight per 24 hours;about 1.0 mg to about 25 mg per kg body weight per 24 hours; about 5.0mg to about 50 mg per kg body weight per 24 hours; about 5.0 mg to about20 mg per kg body weight per 24 hours; about 5.0 mg to about 15 mg perkg body weight per 24 hours.

Alternatively, an effective dosage may be up to about 500 mg/m².Generally, an effective dosage is expected to be in the range of about25 to about 500 mg/m², preferably about 25 to about 350 mg/m², morepreferably about 25 to about 300 mg/m², still more preferably about 25to about 250 mg/m², even more preferably about 50 to about 250 mg/m²,and still even more preferably about 75 to about 150 mg/m².

Typically, in therapeutic applications, the treatment would be for theduration of the disease state.

Further, it will be apparent to one of ordinary skill in the art thatthe optimal quantity and spacing of individual dosages will bedetermined by the nature and extent of the disease state being treated,the form, route and site of administration, and the nature of theparticular individual being treated. Also, such optimum conditions canbe determined by conventional techniques.

It will also be apparent to one of ordinary skill in the art that theoptimal course of treatment, such as, the number of doses of thecomposition given per day for a defined number of days, can beascertained by those skilled in the art using conventional course oftreatment determination tests.

In general, suitable compositions may be prepared according to methodswhich are known to those of ordinary skill in the art and accordinglymay include a pharmaceutically acceptable carrier, diluent and/oradjuvant.

These compositions can be administered by standard routes. In general,the compositions may be administered by the parenteral (e.g.,intravenous, intraspinal, subcutaneous or intramuscular), oral ortopical route. More preferably administration is by the parenteralroute.

The carriers, diluents and adjuvants must be “acceptable” in terms ofbeing compatible with the other ingredients of the composition, and notdeleterious to the recipient thereof.

Examples of pharmaceutically acceptable carriers or diluents aredemineralised or distilled water; saline solution; vegetable based oilssuch as peanut oil, safflower oil, olive oil, cottonseed oil, maize oil,sesame oils such as peanut oil, safflower oil, olive oil, cottonseedoil, maize oil, sesame oil, arachis oil or coconut oil; silicone oils,including polysiloxanes, such as methyl polysiloxane, phenylpolysiloxane and methylphenyl polysolpoxane; volatile silicones; mineraloils such as liquid paraffin, soft paraffin or squalane; cellulosederivatives such as methyl cellulose, ethyl cellulose,carboxymethylcellulose, sodium carboxymethylcellulose orhydroxypropylmethylcellulose; lower alkanols, for example ethanol oriso-propanol; lower aralkanols; lower polyalkylene glycols or loweralkylene glycols, for example polyethylene glycol, polypropylene glycol,ethylene glycol, propylene glycol, 1,3-butylene glycol or glycerin;fatty acid esters such as isopropyl palmitate, isopropyl myristate orethyl oleate; polyvinylpyrridone; agar; carrageenan; gum tragacanth orgum acacia, and petroleum jelly. Typically, the carrier or carriers willform from 10% to 99.9% by weight of the compositions.

The compositions of the invention may be in a form suitable foradministration by injection, in the form of a formulation suitable fororal ingestion (such as capsules, tablets, caplets, elixirs, forexample), in the form of an ointment, cream or lotion suitable fortopical administration, in a form suitable for delivery as an eye drop,in an aerosol form suitable for administration by inhalation, such as byintranasal inhalation or oral inhalation, in a form suitable forparenteral administration, that is, subcutaneous, intramuscular orintravenous injection.

For administration as an injectable solution or suspension, non-toxicparenterally acceptable diluents or carriers can include, Ringer'ssolution, isotonic saline, phosphate buffered saline, ethanol and1,2propylene glycol.

Some examples of suitable carriers, diluents, excipients and adjuvantsfor oral use include peanut oil, liquid paraffin, sodiumcarboxymethylcellulose, methylcellulose, sodium alginate, gum acacia,gum tragacanth, dextrose, sucrose, sorbitol, mannitol, gelatine andlecithin. In addition these oral formulations may contain suitableflavouring and colourings agents. When used in capsule form the capsulesmay be coated with compounds such as glyceryl monostearate or glyceryldistearate which delay disintegration.

Adjuvants typically include emollients, emulsifiers, thickening agents,preservatives, bactericides and buffering agents.

Solid forms for oral administration may contain binders acceptable inhuman and veterinary pharmaceutical practice, sweeteners, disintegratingagents, diluents, flavourings, coating agents, preservatives, lubricantsand/or time delay agents. Suitable binders include gum acacia, gelatine,corn starch, gum tragacanth, sodium alginate, carboxymethylcellulose orpolyethylene glycol. Suitable sweeteners include sucrose, lactose,glucose, aspartame or saccharine. Suitable disintegrating agents includecorn starch, methylcellulose, polyvinylpyrrolidone, guar gum, xanthangum, bentonite, alginic acid or agar. Suitable diluents include lactose,sorbitol, mannitol, dextrose, kaolin, cellulose, calcium carbonate,calcium silicate or dicalcium phosphate. Suitable flavouring agentsinclude peppermint oil, oil of wintergreen, cherry, orange or raspberryflavouring. Suitable coating agents include polymers or copolymers ofacrylic acid and/or methacrylic acid and/or their esters, waxes, fattyalcohols, zein, shellac or gluten. Suitable preservatives include sodiumbenzoate, vitamin E, alpha-tocopherol, ascorbic acid, methyl paraben,propyl paraben or sodium bisulphite. Suitable lubricants includemagnesium stearate, stearic acid, sodium oleate, sodium chloride ortalc. Suitable time delay agents include glyceryl monostearate orglyceryl distearate.

Liquid forms for oral administration may contain, in addition to theabove agents, a liquid carrier. Suitable liquid carriers include water,oils such as olive oil, peanut oil, sesame oil, sunflower oil, saffloweroil, arachis oil, coconut oil, liquid paraffin, ethylene glycol,propylene glycol, polyethylene glycol, ethanol, propanol, isopropanol,glycerol, fatty alcohols, triglycerides or mixtures thereof.

Suspensions for oral administration may further comprise dispersingagents and/or suspending agents. Suitable suspending agents includesodium carboxymethylcellulose, methylcellulose,hydroxypropylmethyl-cellulose, poly-vinyl-pyrrolidone, sodium alginateor acetyl alcohol. Suitable dispersing agents include lecithin,polyoxyethylene esters of fatty acids such as stearic acid,polyoxyethylene sorbitol mono- or di-oleate, -stearate or -laurate,polyoxyethylene sorbitan mono- or di-oleate, -stearate or -laurate andthe like.

The emulsions for oral administration may further comprise one or moreemulsifying agents. Suitable emulsifying agents include dispersingagents as exemplified above or natural gums such as guar gum, gum acaciaor gum tragacanth.

Methods for preparing parenterally administrable compositions areapparent to those skilled in the art, and are described in more detailin, for example, Remington's Pharmaceutical Science, 15th ed., MackPublishing Company, Easton, Pa., hereby incorporated by referenceherein.

The topical formulations of the present invention, comprise an activeingredient together with one or more acceptable carriers, and optionallyany other therapeutic ingredients. Formulations suitable for topicaladministration include liquid or semi-liquid preparations suitable forpenetration through the skin to the site of where treatment is required,such as liniments, lotions, creams, ointments or pastes, and dropssuitable for administration to the eye, ear or nose.

Drops according to the present invention may comprise sterile aqueous oroily solutions or suspensions. These may be prepared by dissolving theactive ingredient in an aqueous solution of a bactericidal and/orfungicidal agent and/or any other suitable preservative, and optionallyincluding a surface active agent. The resulting solution may then beclarified by filtration, transferred to a suitable container andsterilised. Sterilisation may be achieved by: autoclaving or maintainingat 90° C.-100° C. for half an hour, or by filtration, followed bytransfer to a container by an aseptic technique. Examples ofbactericidal and fungicidal agents suitable for inclusion in the dropsare phenylmercuric nitrate or acetate (0.002%), benzalkonium chloride(0.01%) and chlorhexidine acetate (0.01%). Suitable solvents for thepreparation of an oily solution include glycerol, diluted alcohol andpropylene glycol.

Lotions according to the present invention include those suitable forapplication to the skin or eye. An eye lotion may comprise a sterileaqueous solution optionally containing a bactericide and may be preparedby methods similar to those described above in relation to thepreparation of drops. Lotions or liniments for application to the skinmay also include an agent to hasten drying and to cool the skin, such asan alcohol or acetone, and/or a moisturiser such as glycerol, or oilsuch as castor oil or arachis oil.

Creams, ointments or pastes according to the present invention aresemi-solid formulations of the active ingredient for externalapplication. They may be made by mixing the active ingredient infinely-divided or powdered form, alone or in solution or suspension inan aqueous or non-aqueous fluid, with a greasy or non-greasy basis. Thebasis may comprise hydrocarbons such as hard, soft or liquid paraffin,glycerol, beeswax, a metallic soap; a mucilage; an oil of natural originsuch as almond, corn, arachis, castor or olive oil; wool fat or itsderivatives, or a fatty acid such as stearic or oleic acid together withan alcohol such as propylene glycol or macrogols.

The composition may incorporate any suitable surfactant such as ananionic, cationic or non-ionic surfactant such as sorbitan esters orpolyoxyethylene derivatives thereof. Suspending agents such as naturalgums, cellulose derivatives or inorganic materials such as silicaceoussilicas, and other ingredients such as lanolin, may also be included.

The compositions may also be administered in the form of liposomes.Liposomes are generally derived from phospholipids or other lipidsubstances, and are formed by mono- or multi-lamellar hydrated liquidcrystals that are dispersed in an aqueous medium. Any non-toxic,physiologically acceptable and metabolisable lipid capable of formingliposomes can be used. The compositions in liposome form may containstabilisers, preservatives, excipients and the like. The preferredlipids are the phospholipids and the phosphatidyl cholines (lecithins),both natural and synthetic. Methods to form liposomes are known in theart, and in relation to this specific reference is made to: Prescott,Ed., Methods in Cell Biology, Volume XIV, Academic Press, New York, N.Y.(1976), p. 33 et seq., the contents of which is incorporated herein byreference.

Kits

In accordance with the present invention, kits containing δ-tryptasepolypeptide, fragment(s) or analogue(s) thereof, or anti-δ tryptaseantibodies may be prepared. Such kits may is be used, for example, todetect the presence of δ-tryptase, or fragments or analogues thereof, ina biological sample. Detection using such kits is useful for a varietyof purposes, including but not limited to disease diagnosis,epidemiological studies and performing screening methods of the presentinvention.

Kits of the present invention comprising one or more anti δ tryptaseantibodies may further comprise one or more control antibodies which donot react with the δ tryptase polypeptides, or fragments or analoguesthereof, of the present invention. Additionally, kits may contain meansfor detecting the binding of an anti δ tryptase antibody to a δ tryptasepolypeptides, or fragments or analogues thereof, of the presentinvention. For example the one or more anti δ tryptase antibodies may beconjugated to a detectable substrate such as a fluorescent, radioactiveor luminescent compound, an enzymatic substrate, or to a second antibodywhich recognizes the anti δ tryptase antibody and is conjugated to adetectable substrate.

Kits according to the present invention may also include othercomponents required to conduct the methods of the present invention,such as buffers and/or diluents. The kits typically include containersfor housing the various components and instructions for using the kitcomponents in the methods of the present invention.

The present invention will now be described with reference to specificexamples, which should not be construed as in any way limiting the scopeof the invention.

EXAMPLES Example 1 Cloning of δ Tryptase cDNA

Sources of RNA.

HMC-1 cells (5×10⁶, a kind gift from Dr J H Butterfield) were lysed in 1ml of TRI REAGENT™ (Sigma-Aldrich, Sydney, Australia), and 0.2 ml ofchloroform added. Following centrifugation, the aqueous phase wastransferred to a fresh tube and 0.5 ml of isopropanol added. The RNApellet was collected by centrifugation, washed with 75% ethanol,dissolved in dH₂O and stored at −80° C. until required.

Total RNA from adult lung, heart, stomach, spleen, skin, colon, fetalheart and fetal lung, and poly(A⁺) RNA isolated from human lung, wereobtained from commercial sources (Invitrogen, Carlsbad, Calif.).

Preparation of cDNA.

First strand cDNAs were generated using the cDNA Cycle® kit (Invitrogen)from total RNA isolated from the HMC-1 cell line and from poly(A⁺) RNAisolated from human lung. 1.5 μg of HMC-1 total RNA (or 300 ng of lungpoly A⁺ mRNA) and 1 μl of oligo (dT) primer were heated at 65° C. for 10min to remove secondary structure. Reverse transcription was performedfor 1 h at 42° C. in a solution containing 1 μl of RNase inhibitor, 4 μlof 5×RT buffer, 1 μl of 100 mM dNTPs, 1 μl of 80 mM sodium pyrophosphateand 0.5 μl AMV reverse transcriptase. The reaction was terminated byincubating the mixture at 95° C. for 2 min, and was then placed on iceimmediately.

PCR Amplification and Cloning of cDNAs.

PCR amplification of first strand cDNA was performed within 2 h of thereverse transcription reaction. Initially a nested PCR approach was usedto amplify cDNAs, using primers designed according to the sequence of agene that we isolated independently and named delta (δ) tryptase (datanot shown), and according to the published sequence of the mMCP-7-likegenes (GenBank accession numbers AF099147 and AF098327) (Pallaoro etal., 1999). Two sets of primers (F1=5′-CCC GTC CTG GCG AGCCCG-3′/R1=5′-CAG TGA CCC AGG TGG ACA C-3′ and F2=5′-AGT GGC CAG GAT GCTGAG C-3′/R2=5′-TTT GGA CAG GAG GGG CTG GCT-3′) were employed to amplifythe initial product, and a single nested primer pair (NF1=5′-GAG CAA GTGGCC CTG GCA-3′/NR1=5′-GGA CAT AGT GGT GGA TCC AG-3′, see FIG. 2A) wasused on the resulting template. In later experiments a single primerpair (F3=5′-TGC AGC AAA CGG GCA TTG TTG-3′, and R3=5′-AAA GCT GTG GCCCGT ATG GAG-3′) was used to amplify δ tryptase cDNAs.

The PCR reactions were carried out with 2.5 units of AmpliTaq Gold™(Perkin Elmer, Branchburg, N.J.) and 2 μl of the reverse transcriptasereaction mixture. The total reaction volume was 50 μl with a finalconcentration of 10 mM Tris/HCl, pH 8.3, 50 mM KCl, 2 mM MgCl₂, 0.2 mMdNTPs, and 0.1 μM of the appropriate 5′ and 3′ primers. After an initialincubation for 5 min at 94° C., samples were subjected to 35 cycles ofPCR (45 s at 95° C., 60 s at 55° C., and 60 s at 72° C.). This wasfollowed by a final extension step of 72° C. for 10 min. PCR products(10 μl) were visualized on a 1% agarose gel. Appropriately-sizedproducts were excised from the gel, purified with QIAquick gelextraction kit (QIAGEN, Chatsworth, Calif.) and ligated into the plasmidvector pCR®2.1-TOPO (Invitrogen). The ligation mixture was then used totransform TOP10 cells (Invitrogen). The transformation mixture wasplated onto LB/agar plates containing Ampicillin (50 μg/ml), and coatedwith X-gal to enable blue/white color selection. Plasmids containing theappropriate sized inserts were screened for the presence of tryptasecDNAs by PCR using the primer set NF1/NR1. Plasmid DNA was then purifiedfrom clones identified in this manner using a commercial kit (Qiagen).Nucleotide sequencing was performed either in-house using an ABI Prism®BigDye Terminator Cycle Sequencing Ready Reaction Kit and an ABI377PRISM DNA Sequencer (PE Applied Biosystems, Foster City, Calif.), or ata core facility (SUPAMAC, Sydney, Australia).

δ Tryptase cDNA Sequences

PCR amplification of the first strand cDNA template from the HMC-1 cellline generated multiple bands, but only reactions using the F1/R1 primerpair resulted in amplification of the expected 832 bp product (FIG. 1A).The correctly sized band was excised and used as a PCR template with thenested primer pair NF1/NR2. The expected 698 bp PCR product wasgenerated (FIG. 1B), excised, and cloned into the pCR2.1 vector.Sequencing of ten clones revealed the presence of two distinct cDNAsthat we have named δI tryptase and δII tryptase (GenBank accessionnumbers AY055427 and AF206664 respectively). The δI tryptase cDNAsequence matched that predicted from the published partial sequence ofthe mMCP-7-like I gene, and the δII tryptase cDNA matched the publishedexonic sequence of the mMCP-7-like II gene (Pallaoro et al., 1999). ThecDNA and putative amino acid sequence of δII tryptase is shown in FIG.2A. The cDNA sequence of δI tryptase (sequence not shown) was identicalto that of δII except for two nucleotide differences; G²¹⁶ (δII cDNA) toA (δI cDNA) (nucleotide numbering starts from the translation initiationcodon), and G²²⁶ (δII cDNA) to A (δI cDNA). Of the nucleotidedifferences described above only the second, G²²⁶ (δII) to A (δI),results in an altered amino acid residue in the putative proteinproducts (Val in δII and Met in δI).

These results confirm that the primary transcript is spliced aspredicted by Pallaoro et al., 1999. The consequence of this is that,compared to other tryptases, the human δ tryptases possess a prematuretranslation termination codon (T⁷⁰⁶AA) at the beginning of exon 6 whichwould result in the translation of a mature enzyme that is 40 aminoacids shorter than the α/β tryptases (FIG. 3). Despite this truncationthe serine protease catalytic triad His⁴⁴ Asp⁹¹ and Ser¹⁹⁴ (initial Metnumbered as position 1), which is an absolute requirement forproteolytic activity in these enzymes, remains intact. A furtherconsequence of this truncation is the complete loss of the residues thatcomprise loop 2, one of seven that form the substrate-binding cleft inthe α/β tryptases (Pereira et al., 1998; Huang et al., 1999).

A search of the dbEST database found no sequence with high similarity toany of the cDNAs cloned in the present study.

Example 2 In vivo Expression of 5 Tryptase mRNA Transcripts

RT-PCR and Real-Time Quantitative (RTQ) RT-PCR

Initially semi-quantitative RT-PCR (with primers F3/R3) was performed toscreen a broad range of total RNA samples isolated from lung, heart,stomach, spleen, skin and colon. The expression of δ tryptase mRNA wasthen quantified using a real-time quantitative (RTQ) RT-PCR approachperformed on a AB7700 Sequence Detection System (PE Applied Biosystems).Reverse transcription was performed using a commercial kit (PerkinElmer). Briefly, 1 μg of total RNA purified from human lung, heart,spleen, stomach, colon, and the HMC-1 cell line were reverse transcribedaccording to the manufacturers instructions. In control experiments,reverse transcriptase was omitted from the reaction mixture to controlfor possible contamination of the sample with genomic template DNA.Total reaction volume was 50 ul.

Oligonucleotide primers (forward primer DF1=GGC CAC AGC TTT CAA ATC GT,reverse primer DR1=GCA GTT AGG TGC CAT TCA CCT T) and a Taqman probeDTP1 (6FAM-CCT GCC AGG GTG ACT CCG GAG GG) were designed using thePrimerExpress software (PE Applied Biosystems) to specifically detectreverse transcribed δ tryptase mRNA, and not the mRNA of other tryptases(see FIG. 2A). Co-amplification of genomic DNA was avoided by locatingthe forward and reverse primers in separate exons and designing theprobe so that it straddled the exon5/exon 6 boundary.

Optimal concentrations and conditions for amplification were determinedusing the plasmid containing the δ tryptase cDNA as template. Thespecificity for δ tryptase was determined by comparing PCR amplificationof δ tryptase, αII tryptase, or βI tryptase cDNA templates. Cyclingconditions were 50° C.-2 mins, 95° C.-10 mins, then 40-45 cycles of 95°C.-15 secs, 60° C.-60 secs.

For determination of mRNA levels, 6 μl of the appropriate RT reactionmixture was added to 12.5 μl of PCR master mix, 2.5 μl of Taqman probe(2.5 μM), 1 μl each of the forward and reverse primers (18 μM each), and2 μl of dH₂O to give a total reaction mixture of 25 μl.

Relative quantitation of δ tryptase mRNA expression in various tissueswas determined by comparing the sample threshold cycle number (C_(T)),to a standard curve constructed with serial log dilutions (10⁻¹ ng to10⁻⁸ ng) of a plasmid containing the δ tryptase cDNA. Relative copynumber was determined using an algorithm (1 ng of plasmid=2.01342×10⁸copies) and then expressed per μg of total RNA.

For each RNA sample, δ tryptase expression was then normalized forβ-actin expression. The relative quantity of δ tryptase mRNA wasdetermined using commercially available Taqman probe and primers (PEApplied Biosystems), and a standard curve constructed by serial dilutionof a plasmid containing the β-actin cDNA. The standard deviation for theresulting δ tryptase:β-actin ratio was determined using the equation:CV_(ratio)=√(CV_(δ tryptase) ²+CV_(β-actin) ²) where CV=SD/X (standarddeviation/mean). Standards were tested in duplicate and samples intriplicate.

Transcript Expression in vivo

Standard RT-PCR analyses revealed that the δ tryptase genes aretranscribed in a wide range of tissues. Correctly-sized ethidiumbromide-staining bands were visible after 30 cycles, when amplified fromRNA isolated from the HMC-1 cell line, lung, heart, stomach, spleen,skin and colon, as well as in fetal lung and heart (data not shown).Sequencing of RT-PCR products amplified from HMC-1, lung and fetal lungconfirmed their identity (data not shown). The lack of contaminatingsequence indicated that no other tryptase transcripts were beingco-amplified.

Abundance of δ Tryptase

Using an RTQ-RTPCR approach, the relative abundance of δ tryptase andβ-actin mRNA in a range of human tissue was determined using log-linearregressions derived from standard curves (representative regressions; δtryptase: y=−3.0918x+12.996, R^(2=0.9516,) β-actin: y=−4.0552x+11.089,R^(2=0.9776,) where y=ng cDNA and x=C_(T)). When normalised for β-actin,δ tryptase was most abundant in the colon and lung, less abundant in theheart and stomach and just detectable in the spleen (FIG. 4).Significant amounts of δ tryptase mRNA was also detected in the HMC-1cell line. No amplification was apparent in the no template control(NTC, FIG. 4), when primers were omitted from the PCR reaction, whenother tryptase cDNAs were used as template in PCR, or when reversetranscriptase was omitted from the RT reactions (data not shown).

Example 3 Generation of an Anti-δ Tryptase Antibody

NZ white rabbits (Institute of Medical and Veterinary Science, GillesPlains, SA, Australia) were immunised with a δ tryptase-specific peptidethat possessed an amino terminal cysteine and the δ tryptase residuesY¹⁶²HTGLHTGHSFQIVRDD¹⁷⁸ conjugated to diphtheria toxin (Mimotopes,Melbourne Australia). The peptide sequence, located in the regiontranslated from exon 5, has only ˜50% identity to the α/β tryptases (seeFIG. 3). A search of protein databases detected no other protein thatshared this epitope.

Anti-δ tryptase antibodies were affinity purified from antisera usingthe peptide Y¹⁶²-D¹⁷⁸ conjugated to thiopropyl sepharose. Thespecificity of the δ tryptase antibody was confirmed by western blot(see below).

Example 4 In vivo Expression of δ Tryptase Polypeptide

Western Blotting

Purified recombinant δ tryptase (˜0.5 μg, see below for details) andrecombinant βII tryptase (˜1 μg, Promega, Madison, Wis.) were separatedon a 10% SDS polyacrylamide gel and transferred to a PVDF membrane.After blocking for 2 hours at room temperature with 5% skim milkpowder/TBS/0.1% Tween 20, membranes were incubated with affinitypurified δ tryptase anti-peptide antibody (1 μg/ml in TBS/0.1% Tween20). Bound primary antibody was detected using a goat anti-rabbitHRP-conjugated second antibody (Biorad, Hercules, Calif.) diluted1/10000 (2 hrs at RT), followed by exposure to an HRP-chemiluminescencesubstrate for 5 mins (Renaissance Enhanced Luminol Reagent, Dupont NEN,Boston, Mass.). The resulting bands were visualised by exposure toBiomax ML photographic film (Kodak, Rochester, N.Y.). Replicate blotswere probed, as described above, with normal rabbit IgG (1 μg/ml), andwith the mouse monoclonal anti-tryptase antibody AA1 diluted 1/200(Dako, Glostrup, Denmark) followed by goat anti-mouse HRP-conjugate.

Western blot analyses indicated that the δ tryptase antibody recognisedrδ tryptase (see below for details) as a single band of less than 30kDa, but not rβII tryptase (FIG. 5A). Conversely, the AA1 antibodydetected rβII tryptase as a major band of greater than 30 kDa, but didnot recognise rδ tryptase (FIG. 5B).

Immunohistochemistry.

Immunohistochemistry was performed on 4 μm serial sections cut fromformalin-fixed and paraffin embedded samples of human lung, colon,stomach, heart, spleen, and rheumatoid synovium. Sections weredeparaffinised, dehydrated, and rinsed in tap water. Antigen retrievalwas performed by incubating the sections with proteinase K (25 μg/ml in0.1M Tris pH 7, 50 mM EDTA) at 37° C. for 30 mins. Sections were thenrinsed with TBS and blocked with 20% normal goat serum/TBS at roomtemperature for 20 mins. Sections were incubated with primary antibodydiluted in TBS/2% BSA (δ tryptase=4 μg/ml overnight at 4° C., normalrabbit IgG=4 μg/ml overnight at 4° C., AA1 anti-tryptase antibody=1/50dilution for 1 hour at room temperature). Sections were then washed 4times for 5 mins in TBS, and then incubated at room temperature for 30mins with the appropriate biotinylated secondary antibody diluted 1/200in TBS/2% BSA: goat anti-rabbit for δ tryptase and normal rabbit IgG,and goat anti-mouse for AA1 tryptase antibody. Sections were washed 4times for 5 mins in TBS, incubated with avidin-conjugated alkalinephosphatase (Vector Laboratories, Burlingame, Calif.) for 30 mins at RT,and then washed 4 times for 5 mins in TBS. The sections were incubatedin the dark for approximately 15 mins with alkaline phosphatasesubstrate (Vector Red, Vector Laboratories), which gives a red reactionproduct in the presence of AP. All incubations were performed in ahumidified chamber. Sections were rinsed in tap water, counterstainedwith haematoxylin for 30 seconds, rinsed in tap water and cover-slippedwith CrystalMount (Biomeda, Foster City, Calif.). Stained sections wereexamined using an Olympus BX-60 microscope and images captured using aSPOT digital camera (Diagnostic Instruments, Sterling Heights, Mich.).

Immunohistochemical analyses revealed that the δ tryptase polypeptide isexpressed in a range of human tissue including colon (FIGS. 6A, B andE), lung, heart, and synovial tissue (data not shown). Nopositive-staining cells were observed in any tissue when the primaryantibody was omitted, or when normal non-immune rabbit Ig was used asthe primary antibody (FIGS. 6C and F). Many of the positive-stainingcells possessed the morphological characteristics of mast cells.Staining of serial sections of colon tissue revealed the presence ofcells that were positive for both δ tryptase (FIG. 6E) and for the α/βtryptases (ie with the AA1 antibody) (FIG. 6D) indicating that some mastcells express δ tryptase. It was noticeable in the colon tissue that thevast majority of δ tryptase-positive cells were in the mucosa,specifically in the lamina propria between the crypts δtryptase-positive cells were virtually absent from the submucosa andmuscle layers.

Example 5 Generation of Active Recombinant δ Tryptase

When compared to the α/β human tryptases, mature human δ tryptase has a40 amino acid C-terminal truncation. To determine whether it is afunctional protease, recombinant δ tryptase was expressed in bacterialcells and tested for the ability to cleave a panel of trypsin-sensitivesubstrates.

The recombinant fusion protein included an N terminal His-patchthioredoxin region (to increase translation efficiency and solubility),an enterokinase (EK) recognition site (to allow activation of thepro-enzyme), the mature delta tryptase sequence, and C-terminal V5 and6×His tags (to aid detection and purification). As the δ tryptase cDNA(GenBank Accession AF206664) used to generate the expression constructdid not include the sequence coding for the beginning of the maturetryptase, the forward primer (5′ CAC CAT GAT TGT TGG GGG GCA GGA GGC CCCCAG GAG CM GTG GCC CTG G 3′) was designed to include this region. Areverse primer (5′GGT GCC ATT CAC CTT GCA 3′) was designed immediately5′ of the stop codon. The resulting PCR fragment was directionallycloned into the pET102D-TOPO vector (Invitrogen), sequenced in bothdirections, and the construct used to transform BL21 DE3 cells.Following the addition of isopropyl-beta-thiogalactopyranoside (IPTG)(0.5 mM final concentration), the bacterial cells were incubated for 6hours at 37° C. while being agitated vigorously. The cells were pelletedby centrifugation and resuspended in lysis buffer (50 mM NaH₂PO₄, 300 mMNaCl, 10 mM imidazole pH 8.0). The lysate was centrifuged to removecellular debris, and the His-tagged recombinant protein purified fromthe supernatant using a Ni—NTA column.

To enable refolding of the protein it was first denatured in 6MGuanidine hydrochloride buffer (100 mM NaH₂PO₄, 10 mM Tris-HCl, 5 mMDTT, 6M GuHCl, pH 8.0) and introduced slowly (3 ml/hr) into refoldingbuffer (50 mM Tris-HCl, pH 7.5, 0.5M NaCl, 10 mM CHAPS, 2 mM DTT) whilestirring gently. The refolded protein was repurified using a Ni—NTAcolumn and dialysed with 20 mM Tris-HCl pH 7.4, 50 mM NaCl, 2 mM CaCl₂overnight.

Using the same approach a recombinant form of αII tryptase was generatedbased on the cDNA we haves cloned (Genbank accession number AF 206665)and that matched the predicted exonic sequence of the all gene reportedby Pallaoro et al., 1999.

The recombinant enzymes were activated proteolytically by incubating therefolded purified protein with recombinant enterokinase (Novagen) for 16hours at 20° C. in a buffer containing 20 mM Tris-HCl pH 7.4, 50 mMNaCl, 2 mM CaCl₂. Following activation, enterokinase was removed fromthe reaction mixture using an enterokinase cleavage capture kit(Novagen).

The enzymatic activity of recombinant δ tryptase was evaluated bytesting its ability to cleave a panel of three trypsin-susceptiblep-nitroanilide (pNA) chromogenic substrates; N-Benzoyl-Pro-Phe-Arg-pNA,D-Ile-Phe-Lys pNA, and N-p-Tosyl-Gly-Pro-Lys 4-pNA (Sigma-Adrich, StLouis, Mo.) and was compared to that of rαII tryptase, and commerciallyavailable native human lung tryptase (ICN, Costa Mesa, Calif.) andrecombinant human lung βII tryptase (Promega, Madison, Wis.). Pro-δtryptase (ie prior to removal of the EK susceptible activation peptide),recombinant EK alone, and buffer alone acted as negative controls.Approximately equal amounts (˜2 μg) of the enzymes were incubated witheach substrate (10 μg) at 37° C. for 2 hrs, in 100 mM HEPES pH7.5 10%glycerol (total reaction volume=50 μl), and then analysed by HPLC usinga reverse-phase column (4.6×50 mM RP18 Xterra, Waters, Bedford, Mass.).In this initial investigation no exogenous heparin was added. Substratecleavage was determined by the detection of new peaks representing theseparated peptide and nitroanilide moieties. The amount of substratecleaved was estimated by measuring the area under the HPLC peak thatcorresponded to the liberated peptide moiety using Delta T Scan softwareversion 2.04 (Delta T Devices Ltd, Cambridge, UK). The retention time ofthe liberated nitroanilide was constant and was experimentallydetermined to be 2.67 mins.

Recombinant δ tryptase was expressed in bacterial cells, purified on ametal chelating column, refolded, and the mature form of the enzymegenerated by EK cleavage. The mature form of the enzyme was recognisedby an anti-peptide antibody as a single band of less than 30 kDa (FIG.5A). rδ tryptase, rαII tryptase, and commercially available rβIItryptase and native lung tryptase were tested for the ability to cleavea panel of three trypsin-susceptible pNA chromogenic substrates. rβIItryptase was able, with different efficiencies, to cleave all threesubstrates (FIG. 7B;N-p-Tosyl-Gly-Pro-Lys>N-Benzoyl-Pro-Phe-Arg-pNA=D-Ile-Phe-Lys pNA) (onlydata for D-Ile-Phe-Lys pNA is shown for all enzymes). Native lungtryptase was able to cleave two of the substrates (FIG. 7C;N-p-Tosyl-Gly-Pro-Lys>D-Ile-Phe-Lys pNA), while rαII tryptase was ableto cleave all three substrates equally, but less efficiently than rβIItryptase (FIG. 7D). rδ tryptase, while ineffective againstN-Benzoyl-Pro-Phe-Arg-pNA or N-p-Tosyl-Gly-Pro-Lys 4-nitroanilide, wasable to cleave D-Ile-Phe-Lys pNA (FIG. 7E). No substrate cleavage wasdetected in the presence of buffer alone (FIG. 7A), pro-rδ tryptase (ienot activated by EK cleavage, FIG. 7F) and EK alone (data not shown).The amount of substrate cleaved by each enzyme was determined byestimating the area under the peaks that represent the liberated peptideportion of the cleaved substrate. The amount of D-Ile-Phe-Lys pNAsubstrate cleaved by rδ tryptase was approximately 12% as much as thatcleaved by rβII tryptase.

Example 6 Cloning of a δ Tryptase Splice Variant

Source of RNA.

HMC-1 cells (5×10⁶, a kind gift from Dr J H Butterfield) were lysed in 1ml of TRI REAGENT™ (Sigma-Aldrich, Sydney, Australia), and 0.2 ml ofchloroform added. Following centrifugation, the aqueous phase wastransferred to a fresh tube and 0.5 ml of isopropanol added. The RNApellet was collected by centrifugation, washed with 75% ethanol,dissolved in dH₂O and stored at −80° C. until required.

Preparation of cDNA.

First strand cDNAs were generated using the cDNA Cycle® kit (Invitrogen)from total RNA isolated from the HMC-1 cell line. 1.5 μg of HMC-1 totalRNA and 1 μl of oligo (dT) primer were heated at 65° C. for 10 min toremove secondary structure. Reverse transcription was performed for 1 hat 42° C. in a solution containing 1 μl of RNase inhibitor, 4 μl of 5×RTbuffer, 1 μl of 100 mM dNTPs, 1 μl of 80 mM sodium pyrophosphate and 0.5μl AMV reverse transcriptase. The reaction was terminated by incubatingthe mixture at 95° C. for 2 min, and was then placed on ice immediately.

PCR Amplification and Cloning of cDNAs.

PCR amplification of first strand cDNA was performed within 2 h of thereverse transcription reaction using the primers F3=5′-TGC AGC AAA CGGGCA TTG TTG-3′, and R3=5′-AAA GCT GTG GCC CGT ATG GAG-3′.

The PCR reactions were carried out with 2.5 units of AmpliTaq GoId™(Perkin Elmer, Branchburg, N.J.) and 2 μl of the reverse transcriptasereaction mixture. The total reaction volume was 50 μl with a finalconcentration of 10 mM Tris/HCl, pH 8.3, 50 mM KCl, 2 mM MgCl₂, 0.2 mMdNTPs, and 0.1 μM of the appropriate 5′ and 3′ primers. After an initialincubation for 5 min at 94° C., samples were subjected to 35 cycles ofPCR (45 s at 95° C., 60 s at 55° C., and 60 s at 72° C.). This wasfollowed by a final extension step of 72° C. for 10 min. PCR products(10 μl) were visualized on a 1% agarose gel. Appropriately-sizedproducts were excised from the gel, purified with QIAquick gelextraction kit (QIAGEN, Chatsworth, Calif.) and ligated into the plasmidvector pCR-II®-TOPO (Invitrogen). The ligation mixture was then used totransform TOP10 cells (Invitrogen). The transformation mixture wasplated onto LB/agar plates containing Ampicillin (50 μg/ml), and coatedwith X-gal to enable blue/white color selection. Plasmids containing theappropriate sized inserts were screened for the presence of tryptasecDNAs by PCR using the primer set F3/R3. Plasmid DNA was then purifiedfrom clones identified in this manner using a commercial kit (Qiagen).Nucleotide sequencing was performed either in-house using an ABI Prism®BigDye Terminator Cycle Sequencing Ready Reaction Kit and an ABI377PRISM DNA Sequencer (PE Applied Biosystems, Foster City, Calif.), or ata core facility (SUPAMAC, Sydney, Australia).

cDNA Sequence

Sequencing revealed that the cloned cDNA was an alternately-splicedversion of the delta tryptase transcript. By comparing the sequence tothat of the full-length transcript and of the gene sequence, it wasdetermined that the alternate pattern of RNA splicing results in atranscript missing the first 27 nucleotides of the region correspondingto exon 4 in the gene. Thus when translated this would result in aprotein product that is 9 amino acids shorter that a protein producttranslated from the full-length transcript. The rest of the proteinwould remain unchanged as there has been no frame-shift.

Example 7 Compositions for Treatment

The suitable compounds and agents identified by the methods of thepresent invention which are used for the treatment or prevention ofdisease states may be administered alone, although it is preferable thatthey be administered as a pharmaceutical composition.

In accordance with the best mode of performing the invention providedherein, specific preferred compositions are outlined below. Thefollowing are to be construed as merely illustrative examples ofcompositions and not as a limitation of the scope of the presentinvention in any way.

Example 7(a) Composition for Parenteral Administration

A composition for intramuscular injection could be prepared to contain 1mL sterile buffered water, and 1 mg of a suitable agent or compound.

Similarly, a composition for intravenous infusion may comprise 250 ml ofsterile Ringer's solution, and 5 mg of a suitable agent or compound.

Example 7(b) Injectable Parenteral Composition

A composition suitable for administration by injection may be preparedby mixing 1% by weight of a suitable agent or compound in 10% by volumepropylene glycol and water. The solution is sterilised by filtration.

Example 7(c) Capsule Composition

A composition of a suitable agent or compound in the form of a capsulemay be prepared by filling a standard two-piece hard gelatin capsulewith 50 mg of the agent or compound, in powdered form, 100 mg oflactose, 35 mg of talc and 10 mg of magnesium stearate.

Example 7(d) Eye Drop Composition

A typical composition for delivery as an eye drop is outlined below:Suitable agent or compound 0.3 g Methyl Hydroxybenzoate 0.005 g PropylHydroxybenzoate 0.06 g Purified Water about to 100.00 ml.

The methyl and propyl hydroxybenzoates are dissolved in 70 ml purifiedwater at 75° C., and the resulting solution is allowed to cool. The asuitable agent or compound is then added, and the solution sterilised byfiltration through a membrane filter (0.22 μm pore size), andaseptically packed into sterile containers.

Example 7(e) Composition for Inhalation Administration

For an aerosol container with a capacity of 20-30 ml: a mixture of 10 mgof a suitable agent or compound with 0.5-0.8% by weight of a lubricatingagent, such as polysorbate 85 or oleic acid, is dispersed in apropellant, such as freon, and put into an appropriate aerosol containerfor either intranasal or oral inhalation administration.

Example 7(f) Ointment Composition

A typical composition for delivery as an ointment includes 1.0 g of asuitable agent or compound, together with white soft paraffin to 100.0g, dispersed to produce a smooth, homogeneous product.

Example 7(g) Topical Cream Composition

A typical composition for delivery as a topical cream is outlined below:Suitable agent or compound 1.0 g Polawax GP 200 25.0 g Lanolin Anhydrous3.0 g White Beeswax 4.5 g Methyl hydroxybenzoate 0.1 g Deionised &sterilised Water to 100.0 g

The polawax, beeswax and lanolin are heated together at 60° C., asolution of methyl hydroxybenzoate is added and homogenisation achievedusing high speed stirring. The temperature is then allowed to fall to50° C. The agent or compound is then added and dispersed throughout, andthe composition is allowed to cool with slow speed stirring.

Example 7(h) Topical Lotion Composition

A typical composition for delivery as a topical lotion is outlinedbelow: Suitable agent or compound 1.2 g Sorbitan Monolaurate 0.8 gPolysorbate 20 0.7 g Cetostearyl Alcohol 1.5 g Glycerin 7.0 g MethylHydroxybenzoate 0.4 g Sterilised Water about to 100.00 ml

The methyl hydroxybenzoate and glycerin are dissolved in 70 ml of thewater at 75° C. The sorbitan monolaurate, polysorbate 20 and cetostearylalcohol are melted together at 75° C. and added to the aqueous solution.The resulting emulsion is homogenised, allowed to cool with continuousstirring and the agent or compound is added as a suspension in theremaining water. The whole suspension is stirred until homogenised.

REFERENCES

-   Caughey, G. H., W. W. Raymond, J. L. Blount, W. T. H. Leola, M.    Pallaoro, P. J Wolters, and G. M. Verghese. 2000. Characterization    of human γ-tryptases, novel members of the chromosome 16p mast cell    tryptase and prostasin gene families. J. Immunol. 164:6566.-   Fields, S. and O. Song. 1989. A novel genetic system to detect    protein-protein interactions. Nature 340:245.-   Flanagan, J. G., and P. Leder. 1990. The kit ligand: a cell surface    molecule altered in steel mutant fibroblasts. Cell 63:185.-   Harris, J. L., A. Niles, K. Burdick, M. Maffitt, B. J. Backes, J. A.    Ellman, I. Kuntz, M. Haak-Frendscho, and C. S. Craik. 2001.    Definition of the extended substrate specificity determinants for    β-tryptases I and II. J. Biol. Chem. 276:24941.-   Huang, C., L. Li, S. A. Krilis, K. Chanasyk, Y. Tang, Z. Li, J. E.    Hunt, and R. L. Stevens. 1999. Human tryptases α and β/II are    functionally distinct, due in part to a single amino acid difference    in one of the surface loops that forms the substrate binding    cleft. J. Biol. Chem. 274:19670.-   Huang C., G. Morales, A. Vagi, K. Chanasyk, M. Ferrazzi, C.    Burklow, W. T. Qiu, E. Feyfant, A. Sali, and R. L. Stevens. 2000.    Formation of enzymatically active, homotypic, and heterotypic    tetramers of mouse mast cell tryptases. Dependence on a conserved    Trp-rich domain on the surface. J. Biol. Chem. 275:351.-   McNeil H. P., D. S. Reynolds, V. Schiller, N. Ghildyal, D. S.    Gurley, K. F. Austen, and R. L. Stevens. 1992. Isolation,    characterization, and transcription of the gene encoding mouse mast    cell protease 7. Proc. Natl. Acad. Sci. USA. 89:11174.-   Min, H. K., N. Kambe, and L. B. Schwartz. 2001. Human mouse mast    cell protease 7-like tryptase genes are pseudogenes. J. Allergy.    Clin. Immunol. 107:315.-   Pallaoro, M., M. S. Fejzo, L. Shayesteh, J. L. Blount, and G. H.    Caughey. 1999 Characterization of genes encoding known and novel    human mast cell tryptases. J. Biol. Chem. 274:3355.-   Pereira, P. J. B., B. A. Bergner, S. Macedo-Ribeiro, R. Huber, G.    Matschiner, H. Fritz, C. P. Sommerhoff, and W. Bode. 1998. Human    beta-tryptase is a ring-like tetramer with active sites facing a    central pore. Nature 392:306.-   Wong G W. Yasuda S. Madhusudhan M S. Li L. Yang Y. Krilis S A.    Sali A. Stevens R L. 2001 Human tryptase epsilon (PRSS22), a new    member of the chromosome 16p13.3 family of human serine proteases    expressed in airway epithelial cells. J. Biol. Chem.    276(52):49169-82.

1. A purified expressed δ tryptase polypeptide or fragment or analoguethereof.
 2. The polypeptide as claimed in claim 1, wherein thepolypeptide is the human δ tryptase polypeptide or a variant generatedby alternative splicing of the primary RNA transcript encoding thepolypeptide.
 3. The polypeptide as claimed in claim 2, wherein thepolypeptide comprises: (a) the amino acid sequence as set forth in SEQID NO:1 or SEQ ID NO:2; or (b) the amino acid sequence as set forth inSEQ ID NO:1 or SEQ ID NO:2 including one or more conservative amino acidsubstitutions. 4-5. (canceled)
 6. The polypeptide of claim 2 wherein thevariant polypeptide comprises: (a) the amino acid sequence as set forthin SEQ ID NO:3; or (b) the amino acid sequence as set forth in SEQ IDNO:3 including one or more conservative amino acid substitutions. 7-8.(canceled)
 9. A method of identifying a compound that interacts with thepolypeptide or fragment or analogue thereof as claimed in claim 1, themethod comprising the steps of: (a) contacting a candidate compound withthe polypeptide or fragment or analogue thereof as claimed in claim 1under conditions suitable to enable interaction of the candidatecompound to the polypeptide or fragment or analogue thereof; and (b)assaying for activity of the polypeptide or fragment or analoguethereof.
 10. The method of claim 9 wherein assaying for activity of thepolypeptide or fragment or analogue thereof comprises adding a labelledsubstrate and measuring a change in the labelled substrate. 11.(canceled)
 12. A method of screening for a compound that modulates theactivity of the polypeptide or fragment or analogue thereof as claimedin claim 1, the method comprising the steps of: (a) contacting thepolypeptide or fragment or analogue thereof as claimed in claim 1 with acandidate compound under conditions suitable to enable interaction ofthe candidate compound to the polypeptide or fragment or analoguethereof; and (b) assaying for activity of the polypeptide or fragment oranalogue thereof.
 13. The method of claim 12 wherein assaying foractivity of the polypeptide or fragment or analogue thereof comprisesadding a labelled substrate and measuring a change in the labelledsubstrate.
 14. The method of claim 12 wherein the modulation of activityis an inhibition of activity of the polypeptide or fragment or analoguethereof.
 15. A method of diagnosing a disease state, or predispositionto a disease state, in a subject, the method comprising the steps of:(a) isolating a biological sample from the subject; and (b) assaying forexpression of the polypeptide or fragment or analogue thereof as claimedin claim 1 in the sample.
 16. The method of claim 15 wherein assayingfor the expression of the polypeptide or fragment or analogue thereofcomprises contacting the biological sample with a compound capable ofinteracting with the polypeptide such that the interaction can bedetected.
 17. The method of claim 16 wherein the compound capable ofinteracting with the polypeptide or fragment or analogue thereof is ananti-δ tryptase antibody.
 18. The method of claim 15 wherein the diseasestate is an inflammatory disease.
 19. The method of claim 18 wherein theinflammatory disease is a mast cell-associated inflammatory disease. 20.The method of claim 19 wherein the inflammatory disease is selected fromthe group consisting of: asthma, allergic rhinitis, urticaria,angioedema, eczematous anaphylaxis, dermatitis such as atopicdermatitis, hyperproliferative skin disease, peptic ulcers, inflammatorybowel disorder, ocular and vernal conjunctivitis, rheumatoid arthritis,and inflammatory skin conditions.
 21. A method of identifying an agentwhich is an inhibitor of mast cell-mediated inflammation, the methodcomprising contacting a cell or cell extract with the agent, determiningwhether there is a change in the activity of a δ tryptase polypeptide orfragment or analogue thereof, and thereby determining whether the agentis an inhibitor of mast cell-mediated inflammation.
 22. The method ofclaim 21 wherein activity of the polypeptide or fragment or analoguethereof is determined by adding a labelled substrate and measuring achange in the labelled substrate.
 23. The method of claim 21 or 22wherein the agent binds to the δ tryptase polypeptide or fragment oranalogue thereof. 24-25. (canceled)
 26. A method for treating orpreventing a disease state in a subject, the method comprisingadministering to the subject a therapeutically effective amount of acompound identified by the method of claim 9 or an agent identified bythe method of claim
 21. 27. The method of claim 26 wherein the diseasestate is an inflammatory disease.
 28. The method of claim 27 wherein theinflammatory disease is a mast cell-associated disease.
 29. The methodof claim 27 wherein the inflammatory disease is selected from the groupconsisting of: asthma, allergic rhinitis, urticaria, angioedema,eczematous anaphylaxis, dermatitis such as atopic dermatitis,hyperproliferative skin disease, peptic ulcers, inflammatory boweldisorder, ocular and vernal conjunctivitis, rheumatoid arthritis, andinflammatory skin conditions.
 30. A method of inhibiting mastcell-mediated inflammation in a subject, the method comprisingadministering to the subject a therapeutically effective amount of anagent identified by the method of claim 21 or a compound identified bythe method of claim
 9. 31-33. (canceled)